The process of motor axonal guidance and the ensuing process of neuromuscular synapse formation are spatially and temporally precisely coordinated, yet how this achieved at the molecular-cellular level is not well understood. One key player in this process is the vertebrate specific gene muscle specific kinase, MuSK, which aligns the path of incoming motor axon with the location of postsynaptic elements to the muscle center. We have compelling evidence that zebrafish unplugged/MuSK binds wnt11r to initiate this process through a dishevelled dependent signaling cascade, thereby restricting growth cones and synaptic prepattern to the muscle center through a mechanism reminiscent of planar cell polarity (PCP). A major implication is that the spatial alignment between presynaptic growth cones and postsynaptic development might be transmitted through a signaling pathway known to position cellular structures or processes at defined positions within the tissue plane. Specifically, unplugged/ MuSK might play a broader than previously anticipated role to organize a common central muscle zone to which pioneering growth cones and the first acetylcholine receptor clusters are restricted. The objective of the studies described here are threefold. (1) Determine the mechanism through which Wnt signals activate unplugged/MuSK function (through functional studies and live cell imaging to examine the cellular localization of unplugged/MuSK in response to Wnt signals). (2) Determine the extent of similarity between 'classical' PCP and unplugged/MuSK downstream signaling (through functional studies and live cell imaging). (3) Lastly, to maximize our understanding of motor axon guidance, it is critical to analyze and clone two newly identified genetic players in this process, turn out and rush hour (through cellular experiments and molecular cloning). These studies are directly relevant to the study of human disease, since genes known to direct axonal growth and synapse formation are implicated in the cause of human disease states and human inherited disorders, and might also play a role in regeneration after nerve injury. PUBLIC HEALTH RELEVANCE: How motor axons navigate without errors over long distances and select their appropriate synaptic muscle targets, is not fully understood. Using genetic studies, live cell imaging and molecular biological studies, this proposal aims to understand how a key regulator gene, muscle specific kinase (MuSK), controls both axonal pathfinding and synapse formation. This is directly relevant to the study of child health and inherited disorders, because genetic defects in axonal pathfinding and synapse formation cause disorders such as horizontal gaze palsy with progressive scoliosis or congenital myasthenic syndrome.